Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In atherogenesis, low density lipoprotein (LDL, diameter 22 nm) accumulates in the extracellular space of the arterial intima in the form of aggregates of lipid droplets (droplet diameter up to 400 nm). Here we studied the effects of various established in vitro LDL modifications on LDL aggregation and fusion. LDL was subjected to vortexing, oxidation by copper ions, proteolysis by alpha-chymotrypsin, lipolysis by sphingomyelinase, and nonenzymatic glycosylation, and was induced to form adducts with malondialdehyde or complexes with anti-apoB-100 antibodies. To assess the amount of enlarged LDL-derived structures formed (due to aggregation or fusion), we measured the turbidity of solutions containing modified LDL, and quantified the proportion of modified LDL that 1) sedimented at low-speed centrifugation (14,000 g), 2) floated at an increased rate at high-speed centrifugation (rate zonal flotation at 285,000 gmax), 3) were excluded in size-exclusion column chromatography (exclusion limit 40 MDa), or 4) failed to enter into 0.5%. Fast Lane agarose gel during electrophoresis. To detect whether particle fusion had contributed to the formation of the enlarged LDL-derived structures, particle morphology was examined using negative staining and thin-section transmission electron microscopy. We found that 1) aggregation was induced by the formation of LDL-antibody complexes, malondialdehyde treatment, and glycosylation of LDL; 2) fusion of LDL was induced by proteolysis of LDL by alpha-chymotrypsin; and 3) aggregation and fusion of LDL were induced by vortexing, oxidation by copper ions, and lipolysis by sphingomyclinase of LDL. The various modifications of LDL differed in their ability to induce aggregation and fusion.
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PMID:Aggregation and fusion of modified low density lipoprotein. 901 15

Subspecies defining the maturation pathway of bovine chymotrypsinogen to alpha-chymotrypsin have been separated in a single chromatographic run by affinity to iminodiacetic acid-Cu(II) [IDA-Cu(II)] immobilized onto Novarose. A major highlight of the elution pattern is that, as maturation proceeds, these subspecies exhibit a correlated increase in affinity toward IDA-Cu(II). This behavior is analyzed by a combination of physicochemical and molecular modeling techniques to assess the contribution of the two histidines present in chymotrypsins, at positions 40 and 57 on the protein surface. Catalytic His-57 features adequate surface accessibility to serve as a ligand to IDA-Cu(II), but its participation is clearly ruled out by specific chemical modification. In contrast, His-40, whose side chain is buried in the crystal structures of both zymogen and mature enzyme, surprisingly proves the most plausible candidate as an electron donor to IDA-Cu(II). This apparent conflict between histidine accessibility and their implication in IDA-Cu(II) recognition has been rationalized on the basis of their flexibility and/or hydrogen-bonding status, with the following outcome. First, histidine constitutes a useful reporter group for subtle protein conformational fluctuations. Second, static accessibility computation alone provides no unequivocal guideline as to whether a protein residue can serve as a ligand. Third, this study is the first to document the occurrence of a screening effect due to hydrogen bonding of an otherwise "accessible" histidine. A significant corollary to this finding would be that the catalytic histidine is rigidly entrapped in a remarkably strong hydrogen-bonding network, a situation that may pertain to mechanistic aspects of catalysis.
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PMID:Residue accessibility, hydrogen bonding, and molecular recognition: metal-chelate probing of active site histidines in chymotrypsins. 918 84

An enzyme generally catalyzes one well defined reaction with high specificity and efficiency. We report here in contrast that the copper protein hemocyanin of the tarantula Eurypelma californicum exhibits two different functions. These occur at the same active site. While hemocyanin usually is an oxygen carrier, its function can be transformed totally to monophenoloxidase and o-diphenoloxidase activity after limited proteolysis with trypsin or chymotrypsin. N-acetyldopamine (NADA) is more effectively oxidized than L-dopa or dopamine. This irreversible functional switch of tarantula hemocyanin function is limited to the two subunits b and c of its seven subunit types. A conserved phenylalanine in the hemocyanin molecule acts as a placeholder for other substrates that are phenylalanine derivatives. The proteolytic cleavage removes an N-terminal fragment, including the critical phenylalanine residue, which opens an entrance for substrates. Therefore no new arrangement of the active site, with its two copper atoms and the mu - eta2:eta2 bound O2 molecule, is necessary to develop the catalytic function.
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PMID:Tarantula hemocyanin shows phenoloxidase activity. 974 64

This study was mainly aimed to investigate the efficacy of trypsin:chymotrypsin to elicit anti-oxidant properties. In our earlier studies it was observed that the enzyme preparation exhibited an anti-inflammatory action as there was a remarkable reduction in oedema formation and tissue destruction. This led to further study on the amount of lipid peroxidation products formed and the levels of enzymatic and non-enzymatic anti-oxidants and relative trace element contents of copper, selenium, iron and zinc during administration of the enzyme preparation. Decreased formation of lipid peroxidation products was observed in treated group in comparison with the untreated group. Higher levels of enzymatic anti-oxidants mainly super oxide dismutase, catalase, glutathione peroxidase and glutathione-s-transferase and non-enzymatic antioxidant namely ceruloplasmin persisted for a longer period of time in the treated group than in the untreated group. No statistical significance was observed in non-enzymatic antioxidants viz. ascorbic acid and tocopherol levels in both the groups. Increased serum copper and selenium levels in the treated group could be related to higher levels of the ceruloplasmin and glutathione peroxidase observed in the treated group. The above studies support the finding that treatment with the enzyme preparation reduced tissue destruction leading to decreased formation of free radicals and subsequent effective scavenging of free radicals by the higher levels of enzymatic and non-enzymatic anti-oxidants.
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PMID:The efficacy of trypsin: chymotrypsin preparation in the reduction of oxidative damage during burn injury. 977 92

HBp17 was purified by Heparin-Copper biaffinity chromatography and HPLC from conditioned medium of A431 cell. The purified HBp17 was digested by staphylococcus urcus V8 protease or chymotrypsin and the heparin-binding fragments were isolated by Heparin-Sepharose. One binding site of peptide mapping is HBp17 residues 110-145 produced by V8. Another one is HBp17 residues 82-143 which were produced by chymotrypsin digestion. Two binding sites of peptide mapping are overlap. Therefore the residues 110-143 of HBp17 are the principle heparin binding site. The basic amino acid cluster in this region may be contribute to the binding of HBp17 to heparin or heparan sulfate proteoglycan on the cell surface and extracellular matrix.
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PMID:Purification of heparin-binding protein HBp17 and identification of HBp17 heparin binding site. 978 42

Chemical glycosylation of bovine alpha-chymotrypsin, by a glucosamine adduct on the carboxyl group, results in the modification of its catalytic activity. The structural alterations of alpha-chymotrypsin resulting from its glycosylation are studied by immobilized metal-ion affinity chromatography (IMAC) and immobilized metal-ion affinity capillary electrophoresis (IMACE). The chemical glycosylation of alpha-chymotrypsin generates two distinct subpopulations of the protein: one which totally loses the initial affinity for IDA-Cu(II) and another which exhibits an increased affinity for the metal chelate ligand.
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PMID:Structure-function relationship in glycosylated alpha-chymotrypsin as probed by IMAC and IMACE. 1044 72

Rapana venosa hemocyanin (Hc) is a giant oxygen-binding protein consisting of different subunits assembled in a hollow cylinder. The polypeptide chain of each subunit is believed to be folded in several oxygen-binding functional units of molecular mass 50 kDa, each containing a binuclear copper active site. Limited proteolysis with alpha-chymotrypsin of native R. venosa hemocyanin allows the separation of three functional proteolytic fragments with molecular masses of approximately 150, 100, and 50 kDa. The functional fragments, purified by combining gel filtration chromatography and ion-exchange FPLC, were analyzed by means of small-angle X-ray scattering (SAXS). The gyration radius of the 50-kDa Rapana Hc fraction (2.4 nm) agrees well with that calculated on the basis of the dimensions determined by X-ray crystallography for one functional unit of Octopus Hc (2.1 nm). Independent shape determination of the 50- and 100-kDa proteolytic fragments yields consistent low-resolution models. Simultaneous fitting of the SAXS data from these fragments provides a higher-resolution model of the 100-kDa species made of two functional units tilted with respect to each other. The model of the 150-kDa proteolytic fragment consistent with the SAXS data displays a linear chain-like aggregation of the 50-kDa functional units. These observations provide valuable information for the reconstruction of the three-dimensional structure of the minimal functional subunit of gastropod hemocyanin in solution. Furthermore, the spatial relationships among the different functional units within the subunit will help in elucidation of the overall quaternary structure of the oligomeric native protein.
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PMID:Low-resolution structure of the proteolytic fragments of the Rapana venosa hemocyanin in solution. 1062 Mar 34

A full-length cDNA of 794 bp encoding a putative copper/zinc-superoxide dismutase (Cu/Zn-SOD) from Pagrus major was cloned by the PCR approach. Nucleotide sequence analysis of this cDNA clone revealed that it comprises a complete open reading frame coding for 154 amino acid residues. The deduced amino acid sequence showed high similarity (53-91%) with the sequences of Cu/Zn-SOD from other species. Computer analysis of the residues required for coordinating copper (His-47, 49, 64, and 121) and zinc (His-64, 72, 81, and Asp-84), as well as the two cysteines (58 and 147) that form a single disulfide bond, were well conserved among all reported Cu/Zn-SOD sequences. To further characterize the Pagrus major Cu/Zn-SOD, the coding region was subcloned into an expression vector, pET-20b(+), and transformed into Escherichia coli BL21(DE3). The expression of the Cu/Zn-SOD was confirmed by enzyme activity stained on a native-gel and purified by Ni(2+)-nitrilotriacetic acid Sepharose superflow. Dimer was the major form of the enzyme in equilibrium. The dimerization of the enzyme was inhibited under acidic pH (below 4.0 or higher than 10.0). The half-life was 8.6 min and the inactivation rate constant (k(d)) was 9.69 x 10(-2) min(-1) at 70 degrees C. The enzyme activity was not significantly affected under 4% SDS or 0.5 M imidazole. The enzyme was resistant to proteolysis by both trypsin and chymotrypsin.
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PMID:Characterization of copper/zinc-superoxide dismutase from Pagrus major cDNA and enzyme stability. 1182 45

Four accessions of the lesser-known legume, Cassia obtusifolia L. (Sickle pod), collected from four different agroclimatic regions of Western Ghats, were evaluated for agrobotanical traits and chemical composition. Among the four accessions, the Keriparai accession had the highest values for plant height (cm), number of flowers per cluster, number of pods per cluster, pod length (cm), seeds per pod, seed weight (g) per pod and seed recovery percentage. Crude protein ranged from 18.56-22.93%, crude lipid was between 5.35-7.40%, crude fiber ranged from 6.83-9.45%, ash content ranged from 5.14-5.83% and carbohydrate varied from 57.00-60.69%. Globulins constituted the bulk of the seed protein as in most legumes. Mineral profiles, viz., sodium, potassium, calcium, magnesium, phosphorus, iron, copper, zinc and manganese ranged from 42.92-84.83, 758.05-1555.79, 559.92-791.72, 456.36-709.47, 629.13-947.79, 8.42-12.35, 0.93-2.06, 10.60-30.04 and 2.12-4.12 mg/100 g seeds flour, respectively. Seed proteins of all accessions exhibited relatively high levels of non-essential and essential amino acids, with the exception of threonine. The in vitro protein digestibility of the legume ranged from 74.66 to 81.44%. Antinutritional substances such as total free phenolics ranged from 0.34-0.66%; tannins were between 0.47-0.60%; L-DOPA content ranged from 0.98-1.34%; trypsin inhibitor activity varied from 11.4-13.5 TIU/mg protein and chymotrypsin inhibitor activity ranged from 10.8-12.3 CIU/mg protein. Phytohemagglutinating activity also was assayed. In conclusion, the accessions of C. obtusifolia, collected from Western Ghats, South India, could serve as a low-cost source of some important nutrients for humans. The antinutritional factors might have little nutritional significance, if the seeds are processed properly.
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PMID:Agrobotanical traits and chemical composition of Cassia obtusifolia L.: a lesser-known legume of the Western Ghats region of South India. 1204 47

A full-length cDNA clone of 744 bp encoding a putative copper/zinc-superoxide dismutase (Cu/Zn-SOD) from lemon (Citrus limon) was cloned by PCR approach. Nucleotide sequence analysis of this cDNA clone revealed that it comprised an open reading frame coding for 152 amino acid residues. The deduced amino acid sequences showed high identity (65-84%) with the sequences of the Cu/Zn-SODs from other plant species. Computer analysis of the residues required for coordinating copper (His-45, -47, -62, and -119) and zinc (His-62, -70, and -79 and Asp-82), as well as the two cysteines (56 and 145) that form a single disulfide bond, showed they were well-conserved among all reported Cu/Zn-SOD sequences in the present study. To further characterize the lemon Cu/Zn-SOD, the coding region was subcloned into an expression vector, pET-20b(+), and transformed into Escherichia coliBL21(DE3). Expression of the Cu/Zn-SOD was confirmed by enzyme activity staining on a native gel and purified by Ni(2+)-nitrilotriacetic acid Sepharose superflow. The purified enzyme showed two active forms (70% monomer and 30% dimer) in equilibrium, and the specific activity was 7 456 units/mg. The activity of the dimer was 65% higher than that of the monomer. The thermal inactivation rate constant K(d) value calculated for the dimer at 90 degrees C was -7.0 x 10(-3) min(-1), and the half-life for inactivation was 99 min. Both activity and forms of the enzyme were affected very little by acidic pH, basic pH, or 4% SDS. The dimeric structure was more resistant to heat and proteolytic attack with trypsin or chymotrypsin compared to the monomeric structure. Imidazole caused the dimer to dissociate into monomers. These studies suggested subunit interaction might be important for enzyme stability.
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PMID:Copper/zinc-superoxide dismutase from lemon cDNA and enzyme stability. 1245 42


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